An Effective Method for Lovastatin Production by Fungi Over Submerged Fermentation

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Solid state fermentation: An effective method for Lovastatin production by fungi over submerged fermentation

Praveen VK and Savitha J*

Department of Microbiology and Biotechnology, Bangalore University, Jnana Bharathi Campus, Bangalore-560056. India.

Accepted 13 March, 2012

The Enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) catalyses conversion of HMG CoA to mevalonate during cholesterol biosynthesis. Lovastatin is used as an anti-cholesterol drug which blocks HMG CoA reductase activity. Lovastatin has been reported to be produced by Submerged Fermentation (SmF) and Solid State Fermentation (SSF) by fungi. Of-late it is used not only as anti- cholesterol drug but as anti-inflammatory agent, cancer cell apoptosis, restoration of renal function, bone disorders treatment; immune-modulatory role is also being investigated. This review provides insight into the different lovastatin production strategies employed by SSF, its advantages over SmF, optimisation parameters, lovastatin genetics etc.

Key words: HMG CoA. Lovastatin, Fungi, SSF, SmF, Pleiotropic.

INTRODUCTION

The World Health Organization estimated that 17.3 antibiotic and other related pharmacological properties million lives were lost in 2008 and an expected 23.6 (Bedford et al., 1995). Noted among the fungal million people will die of cardiovascular diseases (CVD) metabolites are statins (anti-cholesterol compounds) that by the year 2030 (WHO, 2011). Close to 80% of mortality are considered as the most important class produced by rates were reported from the lower and middle income the polyketide pathway. Statins comprises of Compactin, countries. Hypercholesterolemia is one of the reasons for lovastatin, pravastatin, simvastatin, rosuvastatin, these deaths. The treatment of the hypercholesterolemia atorvastatin and fluvastatin. Compactin and lovastatin are is targeted by decreasing the low density lipoprotein of biological origin whereas simvastatin, pravastatin, (LDL) cholesterol and is best achieved by medications rosuvastatin etc are chemical modifications of compactin when diet and exercise fail (Lloyd-Jones et al.,2009) . A and lovastatin (Chakravarti and Sahai, 2004). The wide variety of biologically active compounds are biosynthetic pathway involved in statin production starts produced by fungi, a large proportion of which are from acetate units linked to each other in a head to- tail produced by the polyketide biosynthetic pathway. Fungal fashion to form a polyketide chain. Lovastatin (Figure 1) polyketides represent structurally diverse group and with prevents formation of mevalonate from HMG Co-A many displaying important biological activities such as (Sreenivasan et al., 2008; Marcin and Stanislaw, 2009) by competitively inhibiting the enzyme HMG CoA Reductase. Lovastatin can exist in two forms i.e hydroxyl from and lactone form, of which the hydroxyl-form is the *Corresponding author. Email: [email protected]. Tel: +91-80- active drug. The lactone forms (e.g. lovastatin, 22961461. Fax: +918023219295 simvastatin, cerivastatin) are lipophilic while the acid

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production of Mevinolin (Lovastatin) from Aspergillus terreus. It was later discovered that in fact what they referred to as lymphomas associated with compactin were actually the accumulation of reductase proteins in endoplasmic reticulum in response to statin therapy., Merck got the approval from Food and Drug Administration (FDA) in 1987 to release Mevinolin into the market. However, Akira Endo in 1989, reported that Monascus sp also produced reductase inhibitors and subsequently obtained a patent (Steilberg, 2006; Endo, 2008).

Figure 1. Structure of Lovastatin Pleiotropic Effects of Lovastatin

The clinical applications of lovastatin have been well forms are hydrophilic for example atorvastatin, documented (Sreenivasan et al., 2008). It finds its fluvastatin, pravastatin (Zamvil and Steinmann., 2002). possible applications for more medical uses such as Lovastatin is obtained from different genera and reducing instances of peripheral vascular diseases, species of filamentous fungi. Several fungal genera prevention of strokes, stabilization of artheromatous including Aspergillus, Penicillium, Monascus, plaques, improved endothelial functions and prevention Paecilomyces, Trichoderma, Scopolariopsis, of thrombus formation (Tandon et al., 2005; Barrios and Doratomyces, Phoma, Phythium, Gymnoascus, Miranda, 2010). Hypomyces and Pleurotus are reported as lovastatin producers (Bizukojc and Stanislaw, 2009, Cabral et al.,

2010, Srinu et al., 2010). In addition even the Alzheimers Disease (AD): Amyloid plaque formation in Basidiomycetes mushrooms have been recently reported brain is noted in patients with AD due to production of to be used. Pleurotus spp and its related strains produce neurotoxic amyloid protein (produced by alternate higher concentrations of lovastatin (Alarcon et al., 2003). processing of amyloid precursor protein). Animal and cell Recently, a marine actinomycete has also been reported culture experiments reported decreased prevalence of to produce lovastatin (Srinu et al., 2010). This review AD on lovastatin treatment (Eckert et al., 2005). Human gives an insight into lovastatin production by SSF in the trials did not yield 100% results and further work needs to last 5-6 years and the possible multiple (pleiotropic be warranted in the same area to attribute exact role of effects) applications of lovastatin in medical field. lovastatin (Eckert et al., 2005).

HISTORY Multiple Sclerosis (MS): Lovastatin supressed production of tumor necreosis factor- α(TNF-α) by A US based company (in 1950’s), Wm. S. Merrell Co., Interferon-α (IFN-α). A decreased level of inflammatory reported a compound with anti-cholesterol property, response and protection of hosts cellular damage was called as Triparanol (MER/ 29) which blocked the noted. Lovastatin also inhibited major histocompatibiliy conversion of desmosterol to cholesterol. Later it was complex-II (MHC-II) upregulation in antigen presenting reported that triparanol was an ineffective drug and cells (APC’s). Atorvastatin prevented or reversed associated with lens cataracts, hair loss in rats and dogs, paralysis in murine experiemental autoimmune also higher doses causes blindness in rats (Steilberg, encephalitis (EAE) models thus indicating the 2006). This led to ban on triparanol. Masao Kuroda and immunomodulatory role of lovastatin (Zamvil and Akira Endo, supported by their team, at Sankyo Co. Steinmann, 2002). Tokyo, screened numerous fungi and discovered that

Penicillium citrinum produced anti-cholesterol compound.

This compound from P. citrinum was christened as Bone Disorders: In murine models prolonged infusions ML236B (referred to as compactin). With several tests or large doses of lovastatin stimulated bone formation the carcinogenic effects of compactin came to light due to both in-vivo and in-vitro (Sreenivasan et al., 2008). Pre- noted lymphomas in dogs treated with higher doses of clinical studies reported that nano particle delivery of compactin. This led to the said company to halt proposed lovastatin may fasten human bone fractures (Garrett et clinical trials. Later, Merck, a competitor, reported the al., 2007). This positive effect may also aid in treatment

Praveen and Savitha 17

of osteoporosis (Sreenivasan et al., 2008). lov D. The multifunctional Polyketide Synthase (PKS) system encodes the LNKS and LDKS in A terreus. LNKS is a gene product of lov B, interacts with lov C (a putative Renal Protection: Glomerulonephritis associated kidney enoyl reductase) to form the dihydro monacolin L which is damage may be retarded by lovastatin treatment. This later converted to Monacolin J. The LDKS, gene product may be possibly due to down regulation of inflammatory of lovF, interacts with lov D (transesterase enzyme) that cytokines and activity of GTPases Ras superfamily. The catalyzes the attachment of the 2-methylbutyric acid to exact role of statins is yet to be elucidated (Buemi et al., monacolin J to form the functional lovastatin (Manzoni 2002). and Rollini, 2002, Sreenivasan et al., 2008, Barrios and Miranda, 2010). In tandem to this works on gene mutation and cloning Cancer: Induction of apoptotic response in human acute of lovastatin genes into other hosts have also been myeloid leukemia (AML) cells was noted on lovastatin reported (Pfeifer and Khosla, 2001, Xie and Tang, 2007). treatment (Xia et al.,2001). Inhibition of The possibility of conversion of Monacolin J (MCJ) to geranylgeranylation of target proteins is the mechanism simvastatin (a semisynthetic form of lovastatin) using lov of lovastatin-induced apoptosis in AML cells. The D gene that has been transferred to E coli has been antiproliferative properties of lovastatin may be used as reported (Xie and Tang, 2007). The substrate used for an effective anticancer drug (Tandon et al., 2005). The the lov D was α-dimethylbutyryl-S-NAC (or α- mechanism underlying lovastatin induced apoptosis of dimethylbutyryl-S-methylthioglycolate). However, this malignant cells remains unclear (Bonovas et al., 2006; reaction resulted in poor product turnout (Xie and Tang, Glynn et al., 2008). About 20 %– 55% reduction in site- 2007). Later, DMB-S-methyl mercaptopropionate (DMB- specific cancers (colorectal, breast, prostate, lung and S-MMP) was used as an alternative substrate. This pancreatic) was observed with the use of statin therapy replacement with a cheaper substrate gave a better (Glynn et al., 2008). Inhibition of Ras farsenylation is simvastatin yield but the rate of conversion was low associated with reduction and proliferation of cancer in (<60%). Lastly, site directed mutagenesis of cysteine to human glioblastoma cells (Xia et al., 2001). Further alanine and aspargine yielded a double mutant of E coli studies need to be carried out to exactly elucidate if with almost >99% transformation of MCJ to simvastatin to statins can be used as anti-cancer drug as concordant yield 18g/L in approximately 18 hrs (Barrios and Miranda, results have not been obtained (Sreenivasan et al., 2010). 2008).

Solid Substrate Fermentation for Lovastatin Rheumatoid Arthritis (RA): Immunomodulation and Production modified endothelial function by lovastatin may aid to decrease problems associated with RA (Sreenivasan et Lovastatin production and optimisation of fermentation al., 2008). parameters has been of great interest since its discovery A recent report suggests the role of statins as an (Kumar et al., 2000, Sayyad et al., 2007, Jaivel and immuno-modulator in treatment of vitiligo (Sreenivasan et Marimuthu, 2010b). Many efforts and trials have been al., 2008)and its beneficial part in graft transplant is being performed to increase the titre (Ferron et al., 2005, Jaivel investigated (Sreenivasan et al., 2008). With its multiple and Marimuthu, 2010a, Prabhakar et al., 2011). Initially, applications, statins in future may play a pivotal role in all production processes were carried in Submerged medico-pharmaceutical field (Tandon et al., 2005). Fermentations (SmF) by varying conditions of its physico- nutritional parameters. The submerged processes have not yielded constant results and higher yield and hence a Biosynthesis and Genetics of Lovastatin Production shift towards to Solid State Fermentation (SSF) was gaining popularity for multiple industrially important The lovastatin pathway initiates from assembly of acetate products such as enzymes, pigments, antibiotics etc. units in head to tail fashion to form the two polyketide SSF has been widely employed in industrial productions chains i.e Lovastatin Nonkedtide Synthase (LNKS) and because of its advantages such as better process control, Lovastatin Diketide Synthase (LDKS) (Barrios et al., maximum substrate utilisation, lower chances of 2010). The first chain is Monacolin J (assembled form 2 contamination, easy downstream processing etc (Pandey acetate units and 9 methionine units) which is later et al., 2001). Many bacteria and fungi have been utilised attached to the second chain i.e 2 Methyl butryl CoA for production of industrially important products by SSF (assembled from 2 acetate units and 1 methionine unit) (Pandey et al., 2001) lovastatin. The genes involved are lov B, lov C, lov F and The production of lovastatin by SmF has given varied

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Table 1. Lovastatin Production by SmF

Lovastatin yield Sl No. Microorganism References (mg/L) 1 Aspergillus terreus 0.40 Szakacs et al., 1998 2 Aspergillus terreus 2200 Kumar et al., 2000 3 Aspergillus terreus 55 Samiee et al., 2003 4 Monascus pilosus 725 Miyake et al., 2006 5 Monascus purpureus 0.318 Sayyad et al., 2007 6 Monascus purpureus 737 Ahmed et al., 2009

results in terms of yield (Table 1). The selection of carbon of SSF over SmF. Numerous data is present regarding and nitrogen source in the growth medium governs the lovastatin production by SmF and optimisation lovastatin yield. Various nutritional combinations have parameters reported. The production of lovastatin by been reported in submerged conditions (Sayyad et al., solid state fermentation (SSF) has gained popularity as it 2007, Sorrentino et al., 2010, Osman et al., 2011,). involves lower media cost, stability of the product, Glucose, lactose, fructose and glycerol have been widely increased yield and better substrate porosity (Chanakya used as carbon source. Glucose and lactose are suited et al., 2011; Reddy et al., 2011). as good carbon source at low concentrations. Higher The substrates used in fermentation include wheat glucose concentration facilitates filamentous growth but bran, rice bran, non-glutinous rice, orange peels, grain decreases titre along with production of ethanol as by- husks (Reddy et al., 2011; Jaivel and Marimuthu, 2010a; product and increasing medium viscosity (Bizukojc and Chankya etal., 2011). Wheat bran topped the list as the Stanislaw, 2009). So a slowly metabolizable carbon best substrate, with a maximum yield of 3273.4 μg/g source like lactose is best suited. Glycerol is also of (Panasuriya and Singhal., 2010) and a yield range of choice (Miyake et al., 2006). Dox medium or modified dox 806 mg/L to 982.3 mg/L (Jaivel and Marimuthu, 2010a). medium also favours lovastatin growth (Atalla et al., Per se, an increase or decrease in moisture content 2008). affects the oxygen and water balance (Panasuriya and Various nitrogen sources often used in organic form Singhal., 2010) and decreases lovastatin yield. The (peptone, soyabean, corn steep liquor) and inorganic higher yield associated with SSF is primarily due to form (ammonium sulphate, ammonium chloride) (Sayyad increased mycelial density provided with an optimum et al., 2007; Atalla et al., 2008) also determine the final moisture range between 60%-70% (Prabhakar et al., yield. 2011). Lovastatin yield of 730 μg/g of dry weight Vis-a-vis there has been use of varied combinations of substrate i.e wheat bran at a moisture level of 65% and carbon: nitrogen sources with different organisms for temperature of 30 OC has been reported using mutated study. Each organism utilizes C:N in varying amounts strain of A terreus (Prabhakar et al., 2011). Rice bran thus leading to varied results (Sayyad et al., 2007, was of good choice specially when supplemented with Sorrentino et al., 2010, Osman et al., 2011). nitrogen source (as rice is poor in nitrogen source) and The addition of supplements to growth medium better compared to rice husk (Pie-Lien et al., 2007). Low influences the yield too. Linoleic acid, butyrolactone, protein content may be dealt by addition of peptone in dodecane, acetic acid favours a higher yield. These substrate. Rice based unsupplemented medium yields supplements work best at low concentrations and work 1.703 mg/g lovastatin using Monascus purpureus MTCC either directly incorporating themselves into specific 369. Rice supplemented with soya bean powder, sucrose pathway or by acting as an enzyme activator (Sorrentino and yeast extract inoculated with M. ruber also resulted in et al., 2010 and Osman et al ., 2011). Butyrolactone and high titre (Xu et al., 2005). Dodecane (2.5%) increased the yield by 3-4 fold Selection of glucose (repressive) along with non- (Bizukojc and Stanislaw, 2009). Sodium acetate repressive carbon source in combination yields increased supplementation triggers better yield by acting as a lovastatin titre in M. pilosus. Glucose and maltose are the precursor for statin synthesis (Osman et al., 2011). best known carbon sources (444 mg/L). Glucose is said Lovastatin production also has been carried by SSF purported to exert repressive action but a combination of approaches with promising results. The results were far glucose, glycerol and peptone in medium is best for M. surprising that expected with high titre of 1110μg/gm dry pilosus to produce lovastatin (Miyake et al., 2006). weight of substrate, thus portraying immense potentials Dextrose, KH2PO 4 and FeSO4 do not aid much in

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lovastatin production when compared to NH4Cl, MgSO4 lovastatin using M purpureus MTCC 369 under SSF. and NaCl when present in medium inoculated with M. Fermentation studies have been revealed the purpureus 369 (Panda et al, 2009b). Various physico- temperature range of 28OC -30OC as the best suited nutritional parameters that govern lovastatin production temperature and at a pH range between 5-6, while for A have been well documented (Sayyad et al., 2007, Marcin fischerii its relative humidity is of 60%, pH 5, and and Stanislaw, 2009, Sorrentino et al., 2010, Osman et temperature set at 300C with lactose and malt extract as al., 2011). its main optimum is best suited for a maximum yield Presence of one amino acid is mandatory in the growth (Chanakya et al. ,2011). SSF holds true potential for media. Riboflavin, pyridoxine and calcium pantothenate production of many industrially important products when used as supplements invariably increased yield, (Pandey et al., 2001). The limitations that may be except for thiamine. Methionine is suited as it is directly encountered in SSF are in controlling of process involved in biosynthetic pathway and gives a yield of 180 parameters and scale up from laboratory to exploitable μg/ml (Marcin and Stanislaw, 2009). It is necessary to industrial level (Mienda et al., 2011). maintain a striking balance between carbon and nitrogen The higher lovastatin production in SSF is primarily source for obtaining a desirable lovastatin production as related to enhanced transcriptional rates of biosynthetic they regulate the biomass and metabolite production genes lov E and lov F resulting in yield increase by 4.6 (Osman et al., 2011). fold and 2 fold respectively. The lov E and lov F Incorporation of various supplements to the growth transcrips accumulation was 20 and 6 fold lower than in medium has been studied (Marcin and Stanislaw, 2009) . SSF when a liquid medium (SmF) of identical Addition of tween-80 increases the yield whereas ZnSO4 concentration when used. Genetically engineered A had no effect on yield, but MgSO4 decreased yield by terreus could synthesise 2,2 dimethyl butyrate (side chain 4.11% (Danuri, 2008). Addition of acetic aci at range of of simvastatin) and produce simvastatin directly rather 0.1% -0.3% also favoured good yield. Glycerol (3%), than lovastatin (Barrios-González et al., 2008). By NaNO3 (0.2%) contributed in higher yield. A glycerol level isolating DNA, RNA and analysing them by above 0.3% decreased titre as it affected fungal cell corresponding blotting techniques it was confirmed that perme-ability (Xu et al., 2005). during SSF the gene transcript levels of lov genes (lov B The use of agro-based wastes (wheat bran, corn hull and lov F) was higher when compared to SmF meanwhile and rice husk), fruit wastes (sugarcane bagasse, orange the expression of gldB (NADP dependent glycerol peel and orange pulp) and their combination has been dehydrogenase), gene for osmotolerance, in SSF seem studied (Prahbakar et al., 2011, Reddy et al., 2011, to indicate the role of osmotic genes in A nidulans when Panasuriya and Singhal., 2010]. Addition of nutrient inert substrate polyurethane foam was used (Barrios- medium to dried substrate has also been the subject of González et al., 2008). This study putatively study with contradictory results. Lower yield of lovastatin substantiates the usage of SSF as a modern optimum is noted when glucose, lactose, sucrose was technological approach for lovastatin production. incorporated into solid substrate (Panasuriya and Singhal, 2010). Glucose, lactose and sucrose when added to Dried Fermented Matter (DFM) tend to CONCLUSION decrease the yield of lovastatin to 2101±51, 2534±29 and 2435±38 μg g- respectively. However, contradictorily a Lovastatin production has gained large scale importance higher yield was recorded when sweet sorghum syrup is with more emphasis on use of SSF approach. This has incorporated with nutrient solution (Jaivel and Marimuthu, resulted in continual search for novel and cheaper 2010a). substrates with stress on optimisation of production Mutation by Ethyl Methyl Sulphonate (EMS) and UV of technology. The molecular level studies also substantiate A terreus KLV28mu21 recorded higher yield (Prabhakar the role of using SSF technology. With the pleiotropic et al., 2011). A terreus isolated from contaminated oyster effects unfolding, statins may be a master key to control mushroom bed subjected to EMS and UV mutation or regulate many major diseases in the future (strains JPM-EMS2and JPM-UV2) produced lovastatin in better appreciable quantity (948.50mg/L and 1553.02 mg/L) (Jaivel and Marimuthu, 2010a). REFERENCES Response Surface Methodology (RSM) approach has Ahmed A, Panda BP, Khan A, Ali M, Javeed S (2009). been well adopted to assess lovastatin yield and Downstreaming and purification of lovastatin from Monascus determine the optimum fermentation parameters (Sayyad O purpureus culture. Thai J. Phar. Sci., 33: 39-46. et al., 2007). A temperature of 29.46 C, fermentation Alarcon J, Aguila S, Arancibia-Avila P, Fuentes O, Zomorano- time of 14.43 days with an initial in-oculum level of 5 ml at Ponce E, Hernandez M (2003). 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